CN116908347A - Method for detecting bacteriostatic agent and flavoring agent of amisulpride oral solution - Google Patents
Method for detecting bacteriostatic agent and flavoring agent of amisulpride oral solution Download PDFInfo
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- CN116908347A CN116908347A CN202310856254.2A CN202310856254A CN116908347A CN 116908347 A CN116908347 A CN 116908347A CN 202310856254 A CN202310856254 A CN 202310856254A CN 116908347 A CN116908347 A CN 116908347A
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- amisulpride
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- flavoring agent
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- 238000000034 method Methods 0.000 title claims abstract description 45
- 229940061256 amisulpride oral solution Drugs 0.000 title claims abstract description 42
- 239000000796 flavoring agent Substances 0.000 title claims abstract description 26
- 239000000022 bacteriostatic agent Substances 0.000 title claims abstract description 22
- 235000013355 food flavoring agent Nutrition 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 44
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 33
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000000243 solution Substances 0.000 claims abstract description 20
- 239000011259 mixed solution Substances 0.000 claims abstract description 14
- 238000010828 elution Methods 0.000 claims abstract description 13
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims abstract description 9
- 239000007924 injection Substances 0.000 claims abstract description 9
- 238000002347 injection Methods 0.000 claims abstract description 9
- 235000019837 monoammonium phosphate Nutrition 0.000 claims abstract description 9
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 4
- 238000005070 sampling Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 abstract description 21
- 238000000926 separation method Methods 0.000 abstract description 16
- 238000002360 preparation method Methods 0.000 abstract description 9
- 239000003814 drug Substances 0.000 abstract description 6
- 238000004458 analytical method Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000008092 positive effect Effects 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 27
- 239000004302 potassium sorbate Substances 0.000 description 27
- 235000010241 potassium sorbate Nutrition 0.000 description 27
- 229940069338 potassium sorbate Drugs 0.000 description 27
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 26
- 239000012535 impurity Substances 0.000 description 23
- 239000012071 phase Substances 0.000 description 23
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 23
- NTJOBXMMWNYJFB-UHFFFAOYSA-N amisulpride Chemical compound CCN1CCCC1CNC(=O)C1=CC(S(=O)(=O)CC)=C(N)C=C1OC NTJOBXMMWNYJFB-UHFFFAOYSA-N 0.000 description 21
- 229960003036 amisulpride Drugs 0.000 description 21
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 18
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 18
- FTLYMKDSHNWQKD-UHFFFAOYSA-N (2,4,5-trichlorophenyl)boronic acid Chemical compound OB(O)C1=CC(Cl)=C(Cl)C=C1Cl FTLYMKDSHNWQKD-UHFFFAOYSA-N 0.000 description 16
- 229960002216 methylparaben Drugs 0.000 description 16
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 16
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 16
- 229940085605 saccharin sodium Drugs 0.000 description 16
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 15
- 239000007788 liquid Substances 0.000 description 15
- 239000000523 sample Substances 0.000 description 13
- 239000000706 filtrate Substances 0.000 description 10
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 9
- 239000002904 solvent Substances 0.000 description 8
- 239000012085 test solution Substances 0.000 description 8
- 238000007865 diluting Methods 0.000 description 6
- 239000006012 monoammonium phosphate Substances 0.000 description 6
- 229960003415 propylparaben Drugs 0.000 description 6
- 238000001914 filtration Methods 0.000 description 5
- 239000013558 reference substance Substances 0.000 description 5
- 239000012488 sample solution Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 4
- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical compound OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- AEQDJSLRWYMAQI-UHFFFAOYSA-N 2,3,9,10-tetramethoxy-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinoline Chemical compound C1CN2CC(C(=C(OC)C=C3)OC)=C3CC2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-UHFFFAOYSA-N 0.000 description 2
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- PHOQVHQSTUBQQK-SQOUGZDYSA-N D-glucono-1,5-lactone Chemical compound OC[C@H]1OC(=O)[C@H](O)[C@@H](O)[C@@H]1O PHOQVHQSTUBQQK-SQOUGZDYSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 235000012209 glucono delta-lactone Nutrition 0.000 description 2
- 229960003681 gluconolactone Drugs 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229940100688 oral solution Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 235000012207 sodium gluconate Nutrition 0.000 description 2
- 239000000176 sodium gluconate Substances 0.000 description 2
- 229940005574 sodium gluconate Drugs 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 1
- 101150049660 DRD2 gene Proteins 0.000 description 1
- 229940121891 Dopamine receptor antagonist Drugs 0.000 description 1
- 101150097070 Drd3 gene Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000561 anti-psychotic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 239000003210 dopamine receptor blocking agent Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 210000003715 limbic system Anatomy 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000008297 liquid dosage form Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 229960003191 potassium methylparaben Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention belongs to the technical field of medicine analysis, and particularly relates to a method for jointly detecting the content of a bacteriostatic agent and a flavoring agent in amisulpride oral solution by using a high performance liquid chromatography. Adopting high performance liquid chromatography, selecting octadecylsilane chemically bonded silica filled reversed phase chromatographic column, taking 0.02mol/L ammonium dihydrogen phosphate solution as mobile phase A, taking acetonitrile and methanol mixed solution as mobile phase B, setting flow rate, column temperature, detection wavelength and sample injection volume, and performing gradient elution. The detection method disclosed by the invention can be used for simultaneously and effectively measuring the content of the 4 substances, has the advantages of simple mobile phase preparation, strong specificity, high separation degree, high sensitivity and good accuracy, greatly improves the separation detection efficiency, provides reliable data reference for the reference reverse direction of amisulpride oral solution and the quality research control of bacteriostat, can be used as a method for measuring the content of the bacteriostat of the amisulpride oral solution in the later period, and has positive effects and practical application values.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a method for jointly detecting the content of a bacteriostatic agent and a flavoring agent in amisulpride oral solution by using a high performance liquid chromatography.
Background
Amisulpride is an aniline substitute type antipsychotic, has the action mechanism of dopamine receptor antagonists, can selectively combine with D2 and D3 dopamine receptors of the central nervous system, blocks dopamine neurons of the limbic system, plays a role in resisting schizophrenia, and has the following structure:
。
the amisulpride oral solution is an oral liquid dosage form of amisulpride, the specification is 100mg/ml, the amisulpride oral solution is not marketed in China, and the amisulpride oral solution is selected as a reference preparation by referring to the announcement public of the chemical imitation pharmaceutical reference preparation catalog (seventeenth lot) and combining the clinical use advantages, wherein the commodity name of the amisulpride is Solian, and the specification is 100mg/ml (package specification is 60 ml).
The reference formulation prescription composition information table shows that the prescription contains a plurality of flavoring agents and bacteriostats. Wherein the flavoring agent comprises saccharin sodium, sodium gluconate and gluconolactone, and the antibacterial agent comprises potassium sorbate, methyl parahydroxybenzoate and propyl parahydroxybenzoate. Due to the nature of each substance, the flavoring agents, namely, gluconolactone and sodium gluconate, have no ultraviolet absorption, cannot be detected by a conventional HPLC method, and need to be studied by developing a separate method. Based on the requirements of product reference reverse and quality control, a detection method capable of effectively detecting the contents of the flavoring agents saccharin sodium, the bacteriostatic agents potassium sorbate, the methylparaben and the propylparaben in the amisulpride oral solution by an HPLC method needs to be developed.
Through searching, the liquid phase method for independently measuring the content of one or two substances of saccharin sodium, potassium sorbate, methyl parahydroxybenzoate and propyl parahydroxybenzoate in amisulpride oral solution is more. The inventor finds that the amisulpride raw material medicine and a plurality of related impurities interfere with detection of 4 additives, and a liquid chromatography method capable of simultaneously measuring the content of the 4 additives in the amisulpride oral solution is lacking in the prior art.
Disclosure of Invention
In order to overcome the defects, the invention aims to provide an HPLC method which can detect saccharin sodium, potassium sorbate, methyl parahydroxybenzoate and propyl parahydroxybenzoate in amisulpride oral solution simultaneously and does not interfere with detection of amisulpride other impurities, and the method has high detection sensitivity, good specificity, accuracy and durability.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention discloses a method for detecting a flavoring agent and a bacteriostatic agent of amisulpride oral solution, which adopts a high performance liquid chromatography, adopts a reversed phase chromatographic column filled with octadecylsilane chemically bonded silica gel, takes 0.02mol/L ammonium dihydrogen phosphate solution as a mobile phase A, takes acetonitrile and methanol mixed solution as a mobile phase B, sets the flow rate, the column temperature, the detection wavelength and the sampling volume, carries out gradient elution, and adopts the following gradient elution procedures:
the main interference factors in the development process of the invention are three: firstly, saccharin sodium and potassium sorbate have larger polarity, have earlier peak time, have approximate retention time as known impurities E, B and F in main component amisulpride, and are easy to be interfered; secondly, the light stability of the potassium sorbate aqueous solution is poor, the potassium sorbate aqueous solution is a liquid preparation, under the illumination condition, potassium sorbate is easy to degrade, and the peak time of the two degradation products is close to that of potassium sorbate, so that the potassium sorbate content detection is easy to be interfered; thirdly, impurities in the amisulpride oral solution have the retention time close to that of the methylparaben, and the content detection of the methylparaben is easy to generate interference. Therefore, the detection method of the invention not only requires that the separation degree of the four substances to be detected meets the requirement, but also requires that amisulpride impurities and potassium sorbate degradation impurities have no interference on the detection of the four substances.
The inventor tries to adopt YMC ODS-AQC 18 (4.6mm×150mm,5 μm) chromatographic column, set A mobile phase as acetonitrile, B mobile phase as 0.02mol/L monoammonium phosphate, and when gradient elution is applied to the content detection of the 4 substances, the known impurities E, B and F in amisulpride are easy to interfere the detection of potassium sorbate and saccharin sodium, and the method can not effectively separate potassium sorbate and the illumination degradation impurities thereof.
According to the embodiment of the invention, the analysis method is determined by optimizing and screening the composition of the mobile phase, the gradient and the spectrum conditions of the column Wen Dengse, and the result shows that the high performance liquid chromatography detection method disclosed by the invention can be used for effectively detecting saccharin sodium, potassium sorbate, methyl p-hydroxybenzoate and propyl p-hydroxybenzoate in amisulpride oral liquid without interfering with detection of other impurities of amisulpride.
According to an embodiment of the invention, the octadecylsilane chemically bonded silica filled reverse phase chromatographic column is selected from Kromasil 100-5-C18 reverse phase chromatographic columns, and has a specification of (100-250) mm× (3.0-5.0) mm, 2-5 [ mu ] m, preferably 150mm×4.6mm,5 [ mu ] m.
According to an embodiment of the invention, the pH of mobile phase a is selected from 5.3 to 5.7, preferably 5.5.
According to an embodiment of the invention, the volume ratio of acetonitrile to methanol in mobile phase B is selected from 5~0:0 to 5, preferably 3:2.
According to an embodiment of the present invention, the detection method disclosed herein gradient elution procedure is preferably selected from:
according to an embodiment of the invention, when the flow rate is selected from 0.9ml/min to 1.1ml/min; the column temperature is 20-35 ℃, the detection wavelength is 254nm, and when the sample volume is 1-100 μl, the peak type and the separation degree of each substance to be detected meet the requirements, and the detection amount of each substance to be detected is basically consistent, which indicates that the method has good durability.
Preferably, the flow rate is selected from 1.0 ml/min; the column temperature was selected from 25℃and the sample volume was selected from 10. Mu.l.
The beneficial effects of the invention are as follows: the method for detecting the content of the amisulpride oral solution has the advantages that the method is developed for the first time, the content of the 1 correctant and the 3 bacteriostat in the amisulpride oral solution can be effectively measured simultaneously, the mobile phase preparation is simple, the specificity is strong, the separation degree is high, the sensitivity is high, the accuracy is good, the separation detection efficiency is greatly improved, reliable data reference is provided for the reference reverse direction of the amisulpride oral solution and the quality research and control of the bacteriostat, and the method can be used as a method for measuring the content of the bacteriostat in the post amisulpride oral solution, and has positive effects and practical application values.
Drawings
FIG. 1 is a high performance liquid chromatogram of a blank solvent for an oral solution of amisulpride of example 1.
FIG. 2 is a high performance liquid chromatogram of the flavor control for the oral amisulpride solution of example 1.
FIG. 3 is a high performance liquid chromatogram of the amisulpride oral solution bacteriostatic agent control of example 1.
FIG. 4 is a high performance liquid chromatogram of a 0 day sample of the amisulpride oral solution of example 1.
FIG. 5 is a high performance liquid chromatogram of a 30-day sample of the oral solution of amisulpride of example 1.
FIG. 6 is a graph showing a comparison of a sample and an impurity mixed solution of amisulpride oral solution of example 1 for 0 day.
FIG. 7 is a high performance liquid chromatogram of a test solution of the oral amisulpride solution of example 2.
FIG. 8 is a high performance liquid chromatogram of a test solution of the amisulpride oral solution of example 3.
FIG. 9 is a high performance liquid chromatogram of a test solution of the amisulpride oral solution of example 4.
Detailed Description
The invention discloses a detection method of a flavoring agent and a bacteriostatic agent in amisulpride oral solution. Those skilled in the art can, with reference to the present disclosure, suitably modify the parameter implementation. It is specifically noted that all similar substitutions and modifications will be apparent to those skilled in the art, and are intended to be included in the present invention. While the detection method of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and suitable modifications and combinations of the method applications described herein can be made to practice and use the techniques of the present invention without departing from the spirit or scope of the invention.
Example 1
The embodiment provides a method for simultaneously measuring the contents of a flavoring agent and a bacteriostatic agent in amisulpride oral solution by using a high performance liquid chromatography, wherein chromatographic conditions are as follows:
chromatographic column: kromasil 100-5-C18 (150 mm. Times.4.6 mm,5 μm)
Mobile phase a:0.02mol/L monoammonium phosphate (triethylamine pH 5.5)
Mobile phase B: acetonitrile-methanol (3:2)
Flow rate: column temperature 1.0 ml/min: detection wavelength at 25 ℃): 254nm sample injection amount: 10 μl of
The gradient elution procedure was:
blank solvent: acetonitrile-water (5:95)
Preparing a test solution: precisely measuring 1.0ml of amisulpride oral solution, placing into a 200ml measuring flask, adding solvent to dissolve and dilute to scale, shaking, filtering, discarding 1ml of primary filtrate, and collecting subsequent filtrate. The illumination influencing factors are prepared by the same method for 30 days.
Flavor control solution: taking a proper amount of saccharin sodium reference substance, precisely weighing, adding a solvent for dissolution and quantitatively diluting to prepare a solution with the concentration of about 160 mug in each 1 ml.
Bacteriostatic agent reference solution: the potassium sorbate, the methylparaben and the propylparaben are taken as the reference substances, respectively, and are precisely weighed, added with 1/3 of the volume of the measuring flask to be dissolved in methanol, and diluted by solvent to prepare a mixed solution which contains about 10 mug of potassium sorbate, 5 mug of methylparaben and 2.5 mug of propylparaben per 1 ml.
Impurity mixed solution: taking the appropriate amounts of the reference substances of the impurity B, the impurity E and the impurity F, precisely weighing, adding methanol for dissolving, and diluting with a solvent to prepare a mixed solution containing about 1 mug of each impurity in each 1 ml.
And precisely measuring 10 mu l of each solution, injecting into a liquid chromatograph, running a gradient program, recording a chromatogram, and carrying out sample injection according to the figures 1-5.
The results show that: the blank solvent in fig. 1 does not interfere with the detection of saccharin sodium, potassium sorbate, methyl parahydroxybenzoate and propyl parahydroxybenzoate, the peak-to-peak type of each substance in the chromatograms of the reference substance solutions in fig. 2 and 3 is good, the number of theoretical plates is greater than 5000, the separation degree between adjacent peaks is greater than 2.0, and the separation condition is good. In the chromatograms of the sample solutions in fig. 4 and 5, the separation degree between the peak of the substance to be detected and the peak of the adjacent impurity is more than 2.0, and the separation is good. In the comparison chart of the impurity mixed solution and the sample solution in fig. 6, all known impurities do not interfere with detection of all substances to be detected, and the method has good specificity and can be used for detecting the contents of the flavoring agent and the bacteriostatic agent in the sample.
Example 2
The chromatographic conditions were as follows:
chromatographic column: kromasil 100-5-C18 (150 mm. Times.4.6 mm,5 μm)
Mobile phase A0.02 mol/monoammonium phosphate (triethylamine pH 5.5)
Mobile phase B: acetonitrile-methanol (3:2)
Flow rate: column temperature 1.0 ml/min: detection wavelength at 25 ℃): 254nm sample injection amount: 10 μl of
The gradient elution procedure was:
preparing a test solution: precisely measuring 1.0ml of amisulpride oral solution, placing into a 200ml measuring flask, adding acetonitrile-water (5:95), dissolving and diluting to scale, shaking, filtering, discarding 1ml of primary filtrate, and collecting subsequent filtrate.
Precisely measuring 10 μl of each solution, injecting into a liquid chromatograph, running gradient program, recording chromatogram, and sampling map as shown in figure 7.
The results show that: in the chromatogram of the sample solution, saccharin sodium, potassium sorbate, amisulpride, methyl parahydroxybenzoate and propyl parahydroxybenzoate sequentially show peaks, and the separation degree between each peak of the substance to be detected and the peak of the adjacent impurity is more than 2.0.
Example 3
The chromatographic conditions were as follows:
chromatographic column: kromasil 100-5-C18 (150 mm. Times.4.6 mm,5 μm)
Mobile phase A0.02 mol/L monoammonium phosphate (triethylamine pH 5.5)
Mobile phase B: acetonitrile-methanol (4:1)
Flow rate: column temperature 1.0 ml/min: detection wavelength at 25 ℃): 254nm sample injection amount: 10 μl of
The gradient elution procedure was:
preparing a test solution: precisely measuring 1.0ml of amisulpride oral solution, placing into a 200ml measuring flask, adding acetonitrile-water (5:95), dissolving and diluting to scale, shaking, filtering, discarding 1ml of primary filtrate, and collecting subsequent filtrate.
Precisely measuring 10 μl of each solution, injecting into a liquid chromatograph, running gradient program, recording chromatogram, and sampling map as shown in figure 8.
The results show that: in the chromatogram of the sample solution, saccharin sodium, potassium sorbate, amisulpride, methyl parahydroxybenzoate and propyl parahydroxybenzoate sequentially show peaks, and the separation degree between each peak of the substance to be detected and the peak of the adjacent impurity is more than 1.9.
Example 4
The chromatographic conditions were as follows:
chromatographic column: kromasil 100-5-C18 (150 mm. Times.4.6 mm,5 μm)
Mobile phase A0.02 mol/L monoammonium phosphate (triethylamine pH 5.5)
Mobile phase B: acetonitrile
Flow rate: column temperature 1.0 ml/min: detection wavelength at 25 ℃): 254nm sample injection amount: 10 μl of
The gradient elution procedure was:
preparing a test solution: precisely measuring 1.0ml of amisulpride oral solution, placing into a 200ml measuring flask, adding acetonitrile-water (5:95), dissolving and diluting to scale, shaking, filtering, discarding 1ml of primary filtrate, and collecting subsequent filtrate.
Precisely measuring 10 μl of each solution, injecting into a liquid chromatograph, running gradient program, recording chromatogram, and sampling with reference to figure 9.
The results show that: in the chromatogram of the sample solution, saccharin sodium, potassium sorbate, amisulpride, methyl parahydroxybenzoate and propyl parahydroxybenzoate sequentially show peaks, and the separation degree between each peak of the substance to be detected and the peak of the adjacent impurity is more than 1.8.
Example 5
The chromatographic conditions were as follows:
chromatographic column: YMC ODS-AQ C18 column (4.6mm.times.250 mm,5 μm)
Mobile phase a:0.02mol/L monoammonium phosphate (triethylamine pH 5.5)
Mobile phase B: acetonitrile
Flow rate: column temperature 1.0 ml/min: wavelength of 35 ℃): 254nm sample injection amount: 10 μl of
Gradient elution:
preparation of the mixed solution: the saccharin sodium, potassium sorbate, methyl hydroxybenzoate and propyl hydroxybenzoate are taken respectively in proper amounts, precisely weighed, dissolved by adding methanol and diluted by acetonitrile-water (5:95) to prepare a solution with about 500 mug in each 1ml, and the proper amounts of the solutions are precisely measured and diluted by acetonitrile-water (5:95) to prepare a mixed solution with about 2 mug in each 1 ml.
Preparation of a raw material medicine solution: proper amount of amisulpride raw material medicine is taken, precisely weighed, dissolved by adding methanol and diluted by acetonitrile-water (5:95) to prepare a solution containing about 0.5mg in each 1 ml.
Precisely measuring 10 μl of each solution, injecting into a liquid chromatograph, running gradient program, and recording chromatogram.
The results show that: in the mixed solution, potassium sorbate, methylparaben and propylparaben sequentially form peaks, and the separation condition between adjacent peaks is good, but the potassium sorbate, the methylparaben and the propylparaben form late peaks, and impurity peaks in the bulk drug interfere detection of the potassium sorbate and the propylparaben, so that the condition is inapplicable.
Example 6
Preparation of a control solution: and respectively taking proper amounts of saccharin sodium, potassium sorbate, methyl parahydroxybenzoate and propyl parahydroxybenzoate as reference substances, precisely weighing, adding methanol for dissolving, and quantitatively diluting with acetonitrile-water (5:95) to prepare a mixed solution containing 160 mug of saccharin sodium, 10 mug of potassium sorbate, 5 mug of methyl parahydroxybenzoate and 2.5 mug of propyl parahydroxybenzoate in each 1 ml.
Preparation of test solution: taking 1.0ml of amisulpride oral solution, placing into a 200ml measuring flask, adding acetonitrile-water (5:95) for dissolution and dilution to scale, shaking uniformly, filtering, discarding 1ml of primary filtrate, and taking the subsequent filtrate.
According to the detection method, specificity, accuracy and durability are verified according to the rule of the analysis method verification guide of the general rule <0512> and the general rule <9101> of the four portions of the Chinese pharmacopoeia 2020 edition, and the results are shown in tables 1-3.
TABLE 1 results of proprietary tests of oral amisulpride solution flavoring and bacteriostatic agent detection methods
The results show that: the impurities in the blank solvent, the negative control and amisulpride do not interfere with the content measurement of the flavoring agent and the bacteriostatic agent of the product, the peak-to-peak type of each substance in the mixed solution and the solution of the test sample is good, the separation degree between the mixed solution and the adjacent impurities is more than 2.0, and the method has good specificity.
TABLE 2 correction of amisulpride oral solution and accuracy test results of bacteriostat detection method
The results show that: and calculating the recovery rate of each substance to be detected according to an external standard method, wherein the recovery rate of each substance is 98.5% -101.0%, and the RSD is less than 1%, so that the method meets the requirements, and shows that the accuracy of the method is good.
TABLE 3 durability test results of amisulpride oral solution flavoring and bacteriostatic agent detection methods
The results show that: when the pH value of the mobile phase A, the proportion of the mobile phase B, the column temperature and chromatographic columns of different brands are slightly changed, the peak type and the separation degree of each substance to be detected meet the requirements, and the detection amounts of the substances to be detected are basically consistent, so that the method has good durability.
The verification result shows that the method can detect the saccharin sodium as the flavoring agent and the potassium sorbate, the methyl parahydroxybenzoate and the propyl parahydroxybenzoate as the bacteriostatic agent in the amisulpride oral solution simultaneously, and effectively control the content of each substance by an external standard method.
Claims (8)
1. A combined detection method of a amisulpride oral solution bacteriostatic agent and a flavoring agent is characterized in that a high performance liquid chromatography is adopted, an octadecylsilane chemically bonded silica filled reversed phase chromatographic column is selected, a 0.02mol/L ammonium dihydrogen phosphate solution is taken as a mobile phase A, a mixed solution of acetonitrile and methanol is taken as a mobile phase B, the flow rate, the column temperature, the detection wavelength and the sampling volume are set, gradient elution is carried out, and the gradient elution procedure is as follows:
。
2. the method for the combined detection of a amisulpride oral solution bacteriostatic agent and a flavoring agent according to claim 1, which is characterized in that the octadecylsilane chemically bonded silica filled reversed phase chromatographic column is selected from Kromasil 100-5-C18 reversed phase chromatographic columns, and the specification is (100-250) mm× (3.0-5.0) mm, 2-5 [ mu ] m, preferably 150mm×4.6mm,5 [ mu ] m.
3. The method for the combined detection of a amisulpride oral solution bacteriostatic agent and a flavoring agent according to claim 1, wherein the pH of the mobile phase A is selected from 5.3-5.7.
4. The method for the combined detection of a amisulpride oral solution bacteriostatic agent and a flavoring agent according to claim 1, which is characterized in that the volume ratio of acetonitrile to methanol in the mobile phase B is selected from 5~0: 0-5.
5. The method for the combined detection of a amisulpride oral solution bacteriostatic agent and a flavoring agent according to claim 4, which is characterized in that the volume ratio of acetonitrile to methanol in the mobile phase B is selected from 3:2.
6. the method for the combined detection of a amisulpride oral solution bacteriostatic agent and a flavoring agent according to claim 1, which is characterized in that the gradient elution procedure is as follows:
。
7. the method for combined detection of amisulpride oral solution bacteriostat and flavoring agent according to claim 1, wherein the flow rate is selected from 0.9 ml/min-1.1 ml/min, the column temperature is selected from 20-35 ℃, the detection wavelength is 254nm, and the sample injection volume is selected from 1-100 μl.
8. The method for combined detection of a bacteriostatic agent and a flavoring agent for amisulpride oral solution according to claim 7, wherein the flow rate is selected from 1.0ml/min, the column temperature is selected from 25 ℃, and the sample injection volume is selected from 10 μl.
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| CN117074579B (en) * | 2023-10-16 | 2023-12-22 | 江苏东科康德药业有限公司 | Analysis method of related substances of amisulpride oral solution |
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